Hydrocortisone (HC) induces an increase in amino acid uptake by human fibroblasts that is 5-fold greater in keloid-derived fibroblasts than in cells from normal dermis. The increase involves primarily System A which preferentially transports proline and glycine. It requires RNA and protein synthesis and results from an elevated Vmax. These findings suggest that HC induces a protein specifically involved in System A amino acid transport. The goal of this research is to determine the molecular basis of the HC-induced increase in amino acid transport in normal and keloid-derived fibroblasts. 1) Membrane proteins from keloid and normal cells will be examined to determine whether there are alterations in amount of HC-induced proteins that correlate with HC-induced increases in System A amino acid transport. Several methods of labeling membrane proteins will be employed to maximize the chance of detecting differences in amount of System A component(s). Other modulators of System A activity that affect normal and keloid-derived cells equally will also be varied to aid in the identification of System A proteins. 2) Plasma membrane vesicles will be prepared to determine whether they retain HC-induced differences in System A activity and protein composition. Use of vesicles increases the chance of identifying System A proteins, due to the partial purification achieved and applicability of additional labeling procedures. 3) Monoclonal antibodies will be prepared to membrane proteins that are induced by HC to a greater extent in keloid-derived fibroblasts than in normal cells. These antibodies will be screened for their ability to differentially bind to the surface of normal and keloid-derived cells grown with and without HC, to bind to purified glucocorticoid-induced proteins, and to alter System A amino acid transport. Detection of amino acid transport proteins in mammalian cells has been hampered by a dearth of mutations that affect transport, and a lack of specific high affinity ligands. Keloids may represent the first example in dermal fibroblasts of a genetic mutation that directly or indirectly alters amino acid transport. In addition to providing that directly or indirectly alters amino acid transport. In addition to providing information important to our understanding of keloids, this research may provide a unique opportunity to study one of the small number of proteins included in the glucocorticoid domain and to isolate and characterize an amino acid transporter from human cells.